Implications of migratory and exotic birds and the mosquito community on West Nile virus transmission.

BACKGROUND: Vector-borne diseases like West Nile virus (WNV) pose a global health challenge, with rising incidence and distribution. Culex mosquitoes are crucial WNV vectors. Avian species composition and bird community diversity, along with vector communities, influence WNV transmission patterns. However, limited knowledge exists on their impact in southwestern Spain, an area with active WNV circulation in wild birds, mosquitoes, and humans. METHODS: To address this, we conducted a comprehensive study investigating the contributions of migratory and exotic bird species to WNV transmission and the influence of mosquito community composition. RESULTS: Analysing 1194 serum samples from 44 avian species, we detected WNV antibodies in 32 samples from 11 species, four for the first time in Europe. Migratory birds had higher WNV exposure likelihood than native and exotic species, and higher phylogenetic diversity in bird communities correlated with lower exposure rates. Moreover, in 5859 female mosquitoes belonging to 12 species, we identified WNV competent vectors like Cx. pipiens s.l. and the Univittatus subgroup. Birds with WNV antibodies were positively associated with competent vector abundance, but negatively with overall mosquito species richness. CONCLUSIONS: These findings highlight the complex interactions between bird species, their phylogenetics, and mosquito vectors in WNV transmission. Understanding these dynamics will help to implement effective disease control strategies in southwestern Spain.

Identification and functional analysis of ABC transporter genes related to deltamethrin resistance in Culex pipiens pallens.

BACKGROUND: Pathogens that reproduce or develop in mosquitoes can transmit several diseases, endanger human health, and overwhelm health systems. Synthetic pyrethroids are the most widely used insecticides against adult mosquitoes, but their widespread use has led to resistance. The adenosine triphosphate (ATP)-binding cassette (ABC) transporters are involved in the resistance monitoring of insects, but their role and underlying mechanisms in insecticide resistance have not been fully elucidated. In the present study, we identified ABC transporter genes in Culex pipiens and investigated their role in the development of insecticide resistance. RESULTS: We identified 63 ABC transporter genes in Cx. pipiens and classified them as per the ABC transporter subfamilies. We also performed phylogenetic analysis. The knockdown rate of the mosquitoes orally fed with the ABC transporter inhibitor verapamil increased after deltamethrin treatment compared with that of the control group. Several genes from the ABCB, ABCC, and ABCG subfamilies were highly expressed in resistant mosquitoes. Immunofluorescence analysis revealed that ABCG6032427 was expressed in the head, chest, abdomen, wings, and legs, and the expression was the highest in the legs. Subsequently, knockdown of ABCG6032427 using RNA interference (RNAi) increased the sensitivity of the mosquitoes to deltamethrin, and scanning and transmission electron microscopy revealed that ABCG6032427 knockdown reduced cuticle thickness and the cuticle became loose and irregular. CONCLUSIONS: ABCG6032427 may modulate cuticle thickness and structure, thus play an important role in the development of deltamethrin resistance in mosquitoes. Thus, it could be a potential target for deltamethrin resistance management in Cx. pipiens. © 2023 Society of Chemical Industry.

Usutu virus: An emerging flavivirus with potential threat to public health in Africa: Nigeria as a case study.

Usutu virus (USUV) is an arthropod-borne virus (arbovirus) of the flaviviridae family (genus Flavivirus) which belong to the Japanese encephalitis virus complex. Culex mosquitoes have been implicated in the transmission of this pathogen. The major susceptible hosts of USUV are migratory birds, thereby potentiating its ability to spread from one region to another globally. Nigeria has the largest economy in Africa with a significant percentage of the gross domestic product relying on the agricultural and animal production industry. This review explores the zoonotic potentials of the virus in Africa, especially Nigeria, with special focus on the devastating sequelae this might lead to in the future if necessary precautionary policies are not enacted and adopted to bolster the surveillance system for mosquito-borne viruses.

Spatiotemporal organization of cryptic North American Culex species along an urbanization gradient.

Landscape heterogeneity creates diverse habitat and resources for mosquito vectors of disease. A consequence may be varied distribution and abundance of vector species over space and time dependent on niche requirements.We tested the hypothesis that landscape heterogeneity driven by urbanization influences the distribution and relative abundance of Culex pipiens, Cx. restuans, and Cx. quinquefasciatus, three vectors of West Nile virus (WNv) in the eastern North American landscape. We collected 9,803 cryptic Culex from urban, suburban, and rural sites in metropolitan Washington, District of Columbia, during the months of June-October, 2019-2021. In 2021, we also collected mosquitoes in April and May to measure early-season abundance and distribution. Molecular techniques were used to identify a subset of collected Culex to species (n = 2,461). Ecological correlates of the spatiotemporal distribution of these cryptic Culex were examined using constrained and unconstrained ordination.Seasonality was not associated with Culex community composition in June-October over three years but introducing April and May data revealed seasonal shifts in community composition in the final year of our study. Culex pipiens were dominant across site types, while Cx. quinquefasciatus were associated with urban environments, and Cx. restuans were associated with rural and suburban sites. All three species rarely coexisted.Our work demonstrates that human-mediated land-use changes influence the distribution and relative abundance of Culex vectors of WNv, even on fine geospatial scales. Site classification, percent impervious surface, distance to city center, and longitude predicted Culex community composition. We documented active Culex months before vector surveillance typically commences in this region, with Culex restuans being most abundant during April and May. Active suppression of Cx. restuans in April and May could reduce early enzootic transmission, delay the seasonal spread of WNv, and thereby reduce overall WNv burden. By June, the highest risk of epizootic spillover of WNv to human hosts may be in suburban areas with high human population density and mixed Culex assemblages that can transmit WNv between birds and humans. Focusing management efforts there may further reduce human disease burden.

Vector Competence of Mosquitoes from Germany for Sindbis Virus.

Transmission of arthropod-borne viruses (arboviruses) are an emerging global health threat in the last few decades. One important arbovirus family is the Togaviridae, including the species Sindbis virus within the genus Alphavirus. Sindbis virus (SINV) is transmitted by mosquitoes, but available data about the role of different mosquito species as potent vectors for SINV are scarce. Therefore, we investigated seven mosquito species, collected from the field in Germany (Ae. koreicus, Ae. geniculatus, Ae. sticticus, Cx. torrentium, Cx. pipiens biotype pipiens) as well as lab strains (Ae. albopictus, Cx. pipiens biotype molestus, Cx. quinquefasciatus), for their vector competence for SINV. Analysis was performed via salivation assay and saliva was titrated to calculate the amount of infectious virus particles per saliva sample. All Culex and Aedes species were able to transmit SINV. Transmission could be detected at all four investigated temperature profiles (of 18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C or 27 ± 5 °C), and no temperature dependency could be observed. The concentration of infectious virus particles per saliva sample was in the same range for all species, which may suggest that all investigated mosquito species are able to transmit SINV in Germany.

Relative Influence of Land Use, Mosquito Abundance, and Bird Communities in Defining West Nile Virus Infection Rates in Culex Mosquito Populations.

Since its introduction to North America in 1999, the West Nile virus (WNV) has resulted in over 50,000 human cases and 2400 deaths. WNV transmission is maintained via mosquito vectors and avian reservoir hosts, yet mosquito and avian infections are not uniform across ecological landscapes. As a result, it remains unclear whether the ecological communities of the vectors or reservoir hosts are more predictive of zoonotic risk at the microhabitat level. We examined this question in central Iowa, representative of the midwestern United States, across a land use gradient consisting of suburban interfaces with natural and agricultural habitats. At eight sites, we captured mosquito abundance data using New Jersey light traps and monitored bird communities using visual and auditory point count surveys. We found that the mosquito minimum infection rate (MIR) was better predicted by metrics of the mosquito community than metrics of the bird community, where sites with higher proportions of Culex pipiens group mosquitoes during late summer (after late July) showed higher MIRs. Bird community metrics did not significantly influence mosquito MIRs across sites. Together, these data suggest that the microhabitat suitability of Culex vector species is of greater importance than avian community composition in driving WNV infection dynamics at the urban and agricultural interface.

St. Louis Encephalitis Virus in the Southwestern United States: A Phylogeographic Case for a Multi-Variant Introduction Event.

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

Surveillance of Culex mosquitoes in Jiangsu Province from 2018 to 2019 / 中国血吸虫病防治杂志

Objective To investigate the distribution and density of Culex mosquito populations and the resistance of Culex pipiens pallens to insecticides in Jiangsu Province in 2018 and 2019. Methods During the period from June to October in 2018 and 2019, six counties (districts, cities) were sampled in southern, northern and central Jiangsu Province as surveillance sites. The density of Culex mosquitoes was measured overnight using the light trapping technique. In addition, Culex pipiens pallens mosquitoes were collected from Hai’an of Nantong City and Yandu District of Yancheng City, central Jiangsu Province, and the sensitivity of female first filial generations to dichlorodiphenyltrichloroethane (DDT), malation, proposur, beta cypermethrin and deltamethrin was tested using the standard WHO insecticide susceptibility test assay. Results A total of 104 423 Culex mosquitoes were captured in six surveillance sites of Jiangsu Province in 2018 and 2019, and Culex quinquefasciatus (49.11%), Culex pipiens pallens (28.38%), and Culex tritaeniorhynchus (21.04%) were predominant species. The density of Culex mosquitoes started to increase since early June, peaked in July and tended to be low in late October. Culex pipiens pallens mosquitoes captured from Hai’an was susceptible to malation, while those from Yandu District were moderately resistant to malation. Culex pipiens pallens mosquitoes from both Yandu and Hai’an were moderately resistant to proposur, and were resistant to DDT, beta cypermethrin and deltamethrin. Conclusions Culex quinquefasciatus, Culex pipiens pallens and Culex tritaeniorhynchus are predominant Culex species in Jiangsu Province. Culex pipiens pallens is resistant to DT, beta cypermethrin and deltamethrin in central Jiangsu Province.

Entomological investigation of an outbreak of Japanese encephalitis in Solan district, Himachal Pradesh.

An outbreak of Japanese encephalitis (JE) was reported in Solan district of Himachal Pradesh, India in May 2018 wherein a total of eight JE cases were reported by the state health department, of which seven cases were confirmed by ELISA. An entomological survey was carried out to investigate the presence of vector mosquitoes in the affected area. Field visits were undertaken in eight villages of two blocks in Solan district. Larval collections were made from the major aquatic sites and emerging mosquito species were identified. Seepage water ditches were the main source of JE vector mosquitoes Culex tritaeniorhynchus and Cx. bitaeniorhynchus. During hand catch collections of adult mosquitoes, three vector species of JE,Culex tritaeniorhynchus (Man Hour Density 1-14), Cx. vishnui (MHD 2) and Cx. bitaeniorhynchus (MHD 1-2) were collected. A few specimens of Cx. tritaeniorhynchus (6 no.) were also collected in light trap collections. Since this was the first reported outbreak of JE from Himachal Pradesh, India, studies on sero-surveillance in addition to bionomics of JE vectors are required for better understanding of epidemiology of JE in Himachal Pradesh. Moreover, there is a need to study the role of climate change especially rising temperature in the context of JE in Himachal Pradesh.

Meta-Transcriptomic Comparison of the RNA Viromes of the Mosquito Vectors Culex pipiens and Culex torrentium in Northern Europe.

Mosquitoes harbor an extensive diversity of 'insect-specific' RNA viruses in addition to those important to human and animal health. However, because most studies of the mosquito virome have been conducted at lower latitudes, little is known about the diversity and evolutionary history of RNA viruses sampled from mosquitoes in northerly regions. Here, we compared the RNA virome of two common northern mosquito species, Culex pipiens and Culex torrentium, collected in south-central Sweden. Following bulk RNA-sequencing (meta-transcriptomics) of 12 libraries, comprising 120 specimens of Cx. pipiens and 150 specimens of Cx. torrentium, we identified 40 viruses (representing 14 virus families) of which 28 were novel based on phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) protein. Hence, we documented similar levels of virome diversity as in mosquitoes sampled from the more biodiverse lower latitudes. Many viruses were also related to those sampled on other continents, indicative of a widespread global movement and/or long host-virus co-evolution. Although the two mosquito species investigated have overlapping geographical distributions and share many viruses, several viruses were only found at a specific location at this scale of sampling, such that local habitat and geography may play an important role in shaping viral diversity in Culex mosquitoes.

Could exotic mosquito-borne diseases emerge in Canada with climate change?

Of the 3,500 species of mosquitoes worldwide, only a small portion carry and transmit the mosquito-borne diseases (MBDs) that cause approximately half a million deaths annually worldwide. The most common exotic MBDs, such as malaria and dengue, are not currently established in Canada, in part because of our relatively harsh climate; however, this situation could evolve with climate change. Mosquitoes native to Canada may become infected with new pathogens and move into new regions within Canada. In addition, new mosquito species may move into Canada from other countries, and these exotic species may bring exotic MBDs as well. With high levels of international travel, including to locations with exotic MBDs, there will be more travel-acquired cases of MBDs. With climate change, there is the potential for exotic mosquito populations to become established in Canada. There is already a small area of Canada where exotic Aedes mosquitoes have become established although, to date, there is no evidence that these carry any exotic (or already endemic) MBDs. The increased risks of spreading MBDs, or introducing exotic MBDs, will need a careful clinical and public health response. Clinicians will need to maintain a high level of awareness of current trends, to promote mosquito bite prevention strategies, and to know the laboratory tests needed for early detection and when to report laboratory results to public health. Public health efforts will need to focus on ongoing active surveillance, public and professional awareness and mosquito control. Canadians need to be aware of the risks of acquiring exotic MBDs while travelling abroad as well as the risk that they could serve as a potential route of introduction for exotic MBDs into Canada when they return home.

Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare.

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.

Assessing human risk of illness with West Nile virus mosquito surveillance data to improve public health preparedness.

Surveillance for West Nile virus (WNV) and other mosquito-borne pathogens involves costly and time-consuming collection and testing of mosquito samples. One difficulty faced by public health personnel is how to interpret mosquito data relative to human risk, thus leading to a failure to fully exploit the information from mosquito testing. The objective of our study was to use the information gained from historic West Nile virus mosquito testing to determine human risk relative to mosquito infection and to assess the usefulness of our mosquito infection forecasting models to give advance warning. We compared weekly mosquito infection rates from 2004 to 2013 to WNV case numbers in Illinois. We then developed a weather-based forecasting model to estimate the WNV mosquito infection rate one to 3 weeks ahead of mosquito testing both statewide and for nine regions of Illinois. We further evaluated human illness risk relative to both the measured and the model-estimated infection rates to provide guidelines for public health messages. We determined that across 10 years, over half of human WNV cases occurred following the 29 (of 210) weeks with the highest mosquito infection rates. The values forecasted by the models can identify those time periods, but model results and data availability varied by region with much stronger results obtained from regions with more mosquito data. The differences among the regions may be related to the amount of surveillance or may be due to diverse landscape characteristics across Illinois. We set the stage for better use of all surveillance options available for WNV and described an approach to modelling that can be expanded to other mosquito-borne illnesses.

Avian malaria infection intensity influences mosquito feeding patterns.

Pathogen-induced host phenotypic changes are widespread phenomena that can dramatically influence host-vector interactions. Enhanced vector attraction to infected hosts has been reported in a variety of host-pathogen systems, and has given rise to the parasite manipulation hypothesis whereby pathogens may adaptively modify host phenotypes to increase transmission from host to host. However, host phenotypic changes do not always favour the transmission of pathogens, as random host choice, reduced host attractiveness and even host avoidance after infection have also been reported. Thus, the effects of hosts' parasitic infections on vector feeding behaviour and on the likelihood of parasite transmission remain unclear. Here, we experimentally tested how host infection status and infection intensity with avian Plasmodium affect mosquito feeding patterns in house sparrows (Passer domesticus). In separate experiments, mosquitoes were allowed to bite pairs containing (i) one infected and one uninfected bird and (ii) two infected birds, one of which treated with the antimalarial drug, primaquine. We found that mosquitoes fed randomly when exposed to both infected and uninfected birds. However, when mosquitoes were exposed only to infected individuals, they preferred to bite the non-treated birds. These results suggest that the malarial parasite load rather than the infection itself plays a key role in mosquito attraction. Our findings partially support the parasite manipulation hypothesis, which probably operates via a reduction in defensive behaviour, and highlights the importance of considering parasite load in studies on host-vector-pathogen interactions.

Avian phenotypic traits related to feeding preferences in two Culex mosquitoes.

Host choice by mosquitoes affects the transmission dynamics of vector-borne infectious diseases. Although asymmetries in mosquito attraction to vertebrate species have been reported, the relative importance of host characteristics in mosquito blood-feeding behavior is still poorly studied. Here, we investigate the relationship between avian phenotypic traits-in particular, morphometry, plumage coloration, and nesting and roosting behavior-and the blood-feeding patterns in two common Culex mosquito species on a North American avian community. Forage ratios of the mosquito species were unrelated to the phylogenetic relationships among bird species. Culex pipiens fed preferably on birds with lighter-colored plumage and longer tarsi; furthermore, solitary roosting avian species were both bitten by Cx. pipiens and Cx. restuans more often than expected. These associations may be explained by greater mosquito attraction towards larger birds with a greater color contrast against the background. Although communally roosting birds may release more cues and attract more mosquitoes, individuals may in fact receive fewer bites due to the encounter-dilution effect. Mosquito feeding behavior is a highly complex phenomenon, and our results may improve understanding of the non-random interaction between birds and mosquitoes in natural communities.

Temporal Variations of Microbiota Associated with the Immature Stages of Two Florida Culex Mosquito Vectors.

Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

Culex mosquitoes are experimentally unable to transmit Zika virus.

We report that two laboratory colonies of Culex quinquefasciatus and Culex pipiens mosquitoes were experimentally unable to transmit ZIKV either up to 21 days post an infectious blood meal or up to 14 days post intrathoracic inoculation. Infectious viral particles were detected in bodies, heads or saliva by a plaque forming unit assay on Vero cells. We therefore consider it unlikely that Culex mosquitoes are involved in the rapid spread of ZIKV.

West Nile virus in Tunisia, 2014: First isolation from mosquitoes.

Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection.

The sequence divergence in cytochrome C oxidase I gene of Culex quinquefasciatus mosquito and its comparison with four other Culex species.

The genetic diversity of Culex quinquefasciatus mosquito based on the standard barcode region of cytochrome C oxidase I (COI) gene fragment was studied in the present study. The COI gene sequences of Cx. quinquefasciatus were also compared with four other species of Genus Culex (i.e. Cx. tritaeniorhynchus, Cx. fuscocephala, Cx. pipiens, and Cx. theileri). Our data set included sequences of Culex mosquitoes from 16 different countries of world. The average intraspecific and interspecific divergences recorded were 0.67% and 8.27%, respectively. The clades for five species were clearly separated except Cx. quinquefasciatus and Cx. pipiens. It is concluded that the DNA barcoding is effective and reliable tool for the identification of selected Culex species but create little problem in case of sister species.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection.

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25 °C as well as 18 °C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25 °C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18 °C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.